Application of CHOP gene in preparation of medicine for treating Alzheimer disease

Abstract

The invention belongs to the technical field of biomedicine, and discloses application of a CHOP gene in preparation of a medicine for treating Alzheimer's disease. According to the application disclosed by the invention, the fact that the beta amyloid deposition of AD can be effectively relieved by knocking out or knocking down the CHOP gene is determined for the first time, neuroinflammation in the brain can be reduced, and the spatial learning and memory ability disorder of AD transgenic mice can be effectively improved. A new target spot is provided for AD treatment, clinical transformation value is achieved, and a preparation with the CHOP gene knocked out or knocked down can be used for preparing the medicine for treating the Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to the application of the CHOP gene in the preparation of drugs for the treatment of Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is a degenerative disease of the central nervous system, characterized by progressive cognitive and behavioral impairments, including symptoms such as memory impairment, aphasia, agnosia, and spatial impairment.

[0003] The pathogenesis of Alzheimer's disease (AD) remains to be fully understood. Current main hypotheses include the Aβ amyloid hypothesis, the Tau protein hypothesis, and the neuroinflammation hypothesis. The Aβ amyloid hypothesis posits that amyloid precursor protein (APP) is abnormally cleaved by β / γ-secretase to generate Aβ42, which aggregates into oligomers and deposits as plaques, triggering neuronal damage. Aβ42 oligomers are the most toxic, inhibiting synaptic mitochondrial function and leading to neuronal injury. The Tau protein hypothesis suggests that hyperphosphorylated Tau protein loses its microtubule stabilizing function, forming intracellular tangles that impair axonal transport and cause neuronal death. The neuroinflammation hypothesis further suggests that Aβ and Tau activate microglia, releasing pro-inflammatory factors such as IL-1β and TNF-α, amplifying neuronal damage. AD is the result of the combined effects of genes, lifestyle, and environmental factors, and is partly caused by specific gene mutations, including those in the APP, PSEN1, PSEN2, APOEε4, and Tau genes. Currently, there is a lack of effective drugs and treatments for AD, and no specific drugs can cure AD or effectively reverse its progression. Therefore, the development of new therapeutic targets for AD is urgently needed. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide the application of the CHOP gene in the preparation of drugs for the treatment of Alzheimer's disease.

[0005] The objective of this invention is achieved through the following technical solution: the application of the CHOP gene in the preparation of drugs for the treatment of Alzheimer's disease. The inventors of this invention discovered that knocking out or knocking down the CHOP gene (C / EBP Homologous Protein, also known as DDIT3 or GADD153) in mice can improve AD symptoms such as β-amyloid protein deposition, improve neuroinflammation in AD, and improve learning and memory abilities.

[0006] Therefore, the application of the CHOP gene in the preparation of drugs for treating Alzheimer's disease is the application of preparations with the CHOP gene knocked out or knocked down in the preparation of drugs for treating Alzheimer's disease.

[0007] The aforementioned knockout or knockdown of the CHOP gene includes small interfering RNA, gene editing molecules, or CHOP antibodies.

[0008] The β-amyloid protein is preferably an insoluble β-amyloid protein from the cerebral cortex and hippocampus.

[0009] The improvement in neuroinflammation in Alzheimer's disease (AD) was achieved by reducing microglia activation. Microglia are significantly activated in the brain, being key participants in AD neuroinflammation. CHOP knockdown / knockout significantly reduced microglia activation, thereby improving neuroinflammation in AD.

[0010] The microglia mentioned are preferably microglia from the cerebral cortex and hippocampus.

[0011] The learning and memory ability mentioned refers to the learning and memory ability of spatial location and direction (spatial positioning).

[0012] In summary, the CHOP knockdown / knockout provided by this invention can effectively alleviate and treat the symptoms of AD, and has the following beneficial effects:

[0013] (1) This invention is the first to clearly demonstrate that knockdown / knockout of the CHOP gene has an ameliorative effect on β-amyloid protein deposition in the AD brain.

[0014] (2) This invention confirms that knockdown / knockout of the CHOP gene has an inhibitory effect on the activation of microglia in the AD brain.

[0015] (3) This invention reveals that knocking down / knockout of the CHOP gene can alleviate the decline in spatial learning and memory ability in aged AD mice, which has extremely important clinical translational value. Attached Figure Description

[0016] Figure 1 This is a schematic diagram illustrating the construction of a transgenic mouse.

[0017] Figure 2 This is a graph showing the expression of the CHOP gene in mouse brain tissue; where * indicates P<0.05 compared with WT mice, and # indicates P<0.05 compared with APP / PS1 mice.

[0018] Figure 3 This is a diagram showing the results of CHOP knockout reducing β-amyloid protein deposition in APP / PS1 mice; in the diagram, β-amyloid protein in the cerebral cortex of 12-month-old APP / PS1 mice (left), APP / PS1 / CHOP+ / - mice (middle), and APP / PS1 / CHOP- / - mice (right) is co-labeled with Thio-S staining (green) and 6E10 antibody (red).

[0019] Figure 4This image shows the results of quantitative detection of β-amyloid protein in the brains of AD mice. A represents the β-amyloid protein levels observed in the brains of 12-month-old APP / PS1, APP / PS1 / CHOP+ / -, and APP / PS1 / CHOP- / - mice using the Thio-S staining method. B represents the quantitative analysis of β-amyloid protein in the cerebral cortex and hippocampus of 9-month-old, 12-month-old, and 15-month-old AD mice. Ctx represents the Cortex (cerebral cortex); Hip represents the Hippocampus; * indicates P < 0.05 compared to APP / PS1 mice.

[0020] Figure 5 The graph shows the detection results of soluble and insoluble forms of Aβ40 and Aβ42 in different mice; where Ctx represents the Cortex (cerebral cortex); Hip represents the Hippocampus; * indicates P<0.05 compared with APP / PS1 mice.

[0021] Figure 6 This is a diagram showing the results of CHOP knockout reducing microglia activation in AD mice; where A is the immunohistochemical staining of brain slices from 9-month-old AD transgenic mice with IbaI antibody; B is the IbaI-positive microglia clustered around Aβ amyloid protein, as observed by IbaI antibody and Thio-S immunofluorescence staining; C is the quantitative statistical result of the positive signal of IbaI immunostaining; * indicates P<0.05 compared with APP / PS1 mice.

[0022] Figure 7 This is a graph showing the results of CHOP knockout improving the spatial learning ability of AD mice; where A is the average time taken by each group of mice to find the platform in the water at different training times, B is the average distance taken by each group of mice to find the platform in the water at different training times, C is the average time taken by each group of mice to find the platform in the water on day 5 of training, and D is the average distance taken by each group of mice to find the platform in the water on day 5 of training; * indicates P<0.05 compared with WT mice, and # indicates P<0.05 compared with APP / PS1 mice.

[0023] Figure 8 This is a graph showing the results of CHOP knockout improving spatial memory in AD mice; where A represents the time each group of mice spent in the quadrant of the platform after the platform was removed, and B represents the number of times they crossed the platform; * indicates P<0.05 compared with WT mice, and # indicates P<0.05 compared with APP / PS1 mice. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0025] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0026] Example 1: Construction of transgenic mice and expression of the CHOP gene in the brain

[0027] Experimental methods:

[0028] 1. Construction of transgenic mice

[0029] The APP / PS1 double transgenic mice used in this study were from the Jackson Laboratory strain B6C3-Tg(APPswe, PSEN1dE9)85Dbo / J; serial number 004462 (purchased from Jackson Laboratory, USA). These mice expressed mutants of human APP and human PS1, with these two gene mutations co-segregating in this mouse population. The CHOP knockout mice (CHOP- / -) were from the Jackson Laboratory strain B6.129S(Cg)-Ddit3tm2.1Dron / J; serial number 005530 (purchased from Jackson Laboratory, USA). APP / PS1 mice were crossed with CHOP- / - mice to obtain two mouse types: WT / CHOP+ / - and APP / PS1 / CHOP+ / -. These two types of mice were then crossed to obtain six mouse types with genotypes of WT, APP / PS1, CHOP+ / -, APP / PS1 / CHOP+ / -, CHOP- / -, and APP / PS1 / CHOP- / - (e.g., Figure 1 (As shown). Truncation of mice was collected 7-12 days after birth. DNA was extracted using a genomic DNA extraction kit and PCR was performed to identify the genotype of each mouse. Identification was performed using primers provided by Jackson Laboratory (as shown below).

[0030] APP / PS1 identification primers:

[0031] Primer 1: 5'-GTG TGATCC ATT CCATCAGC-3';

[0032] Primer 2: 5'-GGATCT CTG AGG GGT CCAGT-3';

[0033] Primer 3: 5'-ATG GTAGAG TAAGCG AGA ACACG-3';

[0034] PCR product bands at 142bp and 265bp indicate APP / PS1 transgenic mice, while PCR product bands at 265bp indicate wild-type mice.

[0035] CHOP identification primers:

[0036] Primer 4: 5'-ATG CCC TTACCT ATC GTG-3';

[0037] Primer 5: 5'-AAC GCC AGG GTT TTC CCAGTC A-3';

[0038] Primer 6: 5'-GCAGGG TCAAGAGTAGTG-3';

[0039] A PCR product band at 544bp indicates WT mice, PCR product bands at 320bp and 544bp indicate CHOP+ / - mice, and PCR product band at 320bp indicates CHOP- / - mice.

[0040] 2. Detection of CHOP gene expression in brain tissue of transgenic mice

[0041] (1) Brain tissue collection: Five mice were raised to 12 months of age in each group. After deep anesthesia, the thoracic cavity was quickly opened to expose the heart, and 20 mL of 0.9% sodium chloride solution was perfused into the heart to remove blood from the brain tissue. The cerebral cortex of each mouse was quickly separated, 1 mL of Trizol was added, and the mice were then flash-frozen in liquid nitrogen.

[0042] (2) Homogenize the tissue, add 0.2 mL of chloroform (20% of the volume of Trizol), shake vigorously for 15 seconds, and let stand at room temperature for 3 minutes. Centrifuge at 4℃ and 12,000×g for 15 minutes.

[0043] (3) Transfer the upper aqueous phase to a new centrifuge tube, add an equal volume of pre-cooled isopropanol, gently invert to mix, and let it precipitate overnight at -20°C. Centrifuge at 4°C and 12,000×g for 10 min.

[0044] (4) Discard the supernatant, add 1 mL of 75% ethanol (prepared with DEPC water), and gently vortex to suspend the precipitate. Centrifuge at 7,500×g for 5 min at 4℃, and discard the supernatant. Dry at room temperature for 5-10 min with the lid off.

[0045] (5) Add 20-50 μL of DEPC water, gently tap the tube wall to dissolve the precipitate, and store at -80℃.

[0046] (6) The RNA concentration was measured, and reverse transcription was performed using a reverse transcription kit to obtain cDNA.

[0047] (7) The levels of CHOP gene and Actin internal reference gene were detected by using a real-time PCR kit (SYBR Green), and the relative expression level of CHOP gene was calculated.

[0048] The primers for the CHOP gene real-time PCR are as follows:

[0049] Primer 7 (upstream primer): 5'-AAG CCT GGT ATG AGG ATC TGC-3';

[0050] Primer 8 (downstream primer): 5'-TTC CTG GGG ATG AGATAT AGG TG-3'.

[0051] The primers for Actin gene real-time PCR are as follows:

[0052] Primer 9 (upstream primer): 5'-GGCTGTATTCCCCTCCATCG-3';

[0053] Primer 10 (downstream primer): 5'-CCAGTTGGTAACAATGCCATGT-3'.

[0054] The reaction system for real-time PCR is as follows:

[0055] Step (6) yields 2 μL of template obtained by diluting cDNA 10 times, 10 μL of 2×SYBR Green qPCR Mix, 0.4 μL of upstream primer at a concentration of 10 μM, 0.4 μL of downstream primer at a concentration of 10 μM, and 2 μL of ddH2O.

[0056] (c) Real-time PCR reaction conditions: 95℃ for 2 min; 95℃ for 15 s, 60℃ for 60 s, 40 cycles.

[0057] (8) The gene expression differences between groups were compared using a relative quantitative algorithm. By comparing the Ct value of the CHOP gene with the Ct value of the Actin internal reference gene, the ΔCt of each group could be obtained. Then, the ΔΔCt of each group was calculated by comparing each group with the wild-type control group, and the fold change of the CHOP gene in each genotype mouse compared with the wild-type mouse was analyzed.

[0058] Experimental results:

[0059] In the cerebral cortex tissue of 12-month-old APP / PS1 mice, CHOP gene expression was significantly increased compared to wild-type mice. However, CHOP gene expression was significantly decreased in APP / PS1 / CHOP+ / - mice, approaching that of wild-type mice. CHOP gene expression was extremely low, almost undetectable, in the brain tissue of CHOP- / - and APP / PS1 / CHOP- / - mice, indicating that the CHOP gene was knocked down / knocked out in the brain tissue of heterozygous CHOP+ / - and homozygous CHOP- / - knockout mice. Figure 2 ).

[0060] Example 2: CHOP knockout reduces the accumulation of β-amyloid protein in AD.

[0061] Experimental methods:

[0062] 1. Sampling, fixation, and sectioning of brain tissue from transgenic mice

[0063] Five to seven 12-month-old APP / PS1, APP / PS1 / CHOP+ / -, and APP / PS1 / CHOP- / - transgenic mice were used in each group for the experiment. The mice were first anesthetized, and after deep anesthesia, the thoracic cavity was quickly opened to expose the heart. The heart was perfused with 50 mL of 0.9% sodium chloride solution, followed by 100 mL of fixative (4% paraformaldehyde solution). Brain tissue was dissected and placed in the fixative for 24 hours, then replaced with 30% sucrose solution. After the brain tissue settled, it was frozen sectioned using a sliding microtome (Lecia). The section thickness was 25 μm, and five brain slices with a bregma of -1.46 to -3.08 were selected for staining and quantification.

[0064] 2. Aβ Immunofluorescence Staining Method

[0065] (1) Brain slices were treated in 0.1% v / v Triton-X 100 at room temperature for 10 minutes, and then washed 3 times with phosphate buffer (137mM NaCl, 2.7mM KCl, 4.3mM Na2HPO4, 1.4mM KH2PO4, pH 7.2-7.4) for 10 minutes each time.

[0066] (2) Block with antibody blocking solution (1% bovine serum albumin) at room temperature for 1 hour;

[0067] (3) The Aβ-specific antibody 6E10 was diluted with 1% bovine serum albumin at a volume ratio of 1:1000. The slides were then transferred to the primary antibody solution and incubated overnight at 4°C. The slides were washed three times with phosphate buffer for 10 minutes each time on the second day.

[0068] (4) Transfer the sections to secondary antibody solution (goat anti-mouse Fluor Alexa 555, 1:1000) and incubate at room temperature for 2 hours. Wash 3 times with phosphate buffer, 10 minutes each time;

[0069] (5) The brain slices are attached to a glass slide that has been pre-coated with gelatin.

[0070] 3. Thio-S staining of β-amyloid plaques in brain tissue sections

[0071] (1) The brain slices are attached to a glass slide that has been pre-coated with gelatin.

[0072] (2) The attached brain slices were stained in 0.05% Thio-S prepared with 50% alcohol for 8 minutes in the dark.

[0073] (3) Place in 80% alcohol for 10 seconds, and repeat once.

[0074] (4) Wash three times with plenty of distilled water.

[0075] (5) Brain slices were incubated at 4°C for more than 30 minutes in a high concentration of phosphate buffer (411mM NaCl, 8.1mM KCl, 30mM Na2HPO4, 5.2mM KH2PO4, pH 7.2).

[0076] (6) Rinse with distilled water.

[0077] (7) Mount the slide with fluorescent mounting medium.

[0078] (8) Quantification of Thio-S staining: Half of the brain slice was photographed using a fluorescence microscope, and then the ThioS positive signals in the cerebral cortex and hippocampus were quantified using ImageJ software. First, the image was converted to 8-bit format. Then, the cerebral cortex and hippocampus were marked out for quantification. The threshold was adjusted to select the ThioS positive signal. The percentage of the total area occupied by the positive signal in the selected area was measured for statistical analysis.

[0079] Experimental results:

[0080] β-amyloid deposition is an important pathological feature in Alzheimer's disease (AD). This invention examined β-amyloid deposition in 12-month-old APP / PS1, APP / PS1 / CHOP+ / -, and APP / PS1 / CHOP- / - mice. First, mouse brain slices were immunohistochemically stained with the Aβ-specific antibody 6E10, and ThioS staining was performed for co-labeling. It was found that these staining agents could co-localize well to β-amyloid plaques in the brain, and compared to age-matched APP / PS1 mice, the accumulation of β-amyloid in the cerebral cortex of APP / PS1 / CHOP+ / - and APP / PS1 / CHOP- / - mice was significantly reduced. Figure 3 Next, Thio-S staining was used to examine β-amyloid plaques in the cerebral cortex and hippocampus of AD mice. Figure 4 A in the image was quantified using ImageJ. Figure 4 (B in the middle). Figure 4 The statistical results of B showed that 9, 12 and 15-month-old APP / PS1 / CHOP+ / - and APP / PS1 / CHOP- / - mice had significantly fewer β-amyloid plaques in both the cerebral cortex and hippocampus than APP / PS1 mice.

[0081] Example 3: CHOP knockout reduces Aβ40 and Aβ42 in the brain tissue of AD mice.

[0082] Experimental methods:

[0083] 1. Brain tissue harvesting: APP / PS1, APP / PS1 / CHOP+ / -, and APP / PS1 / CHOP- / - transgenic mice, aged 12 months, were fed in groups of 8-10. After deep anesthesia, the thoracic cavity was rapidly opened to expose the heart, which was then perfused with 20 ml of 0.9% sodium chloride solution to remove blood from the brain tissue. The cerebral cortex and hippocampus of each mouse were quickly separated, weighed, and flash-frozen in liquid nitrogen.

[0084] 2. Sample preparation: Add RIPA buffer to the tissue at a mass (mg): volume (μL) ratio of 1:9 and homogenize on ice. Lyse on ice for 30 minutes, shaking occasionally. Centrifuge at 100,000g, 4℃ for 60 minutes, and collect the supernatant (the RIPA-soluble fraction) to determine the soluble forms of Aβ40 and Aβ42. Add an equal volume of 5M guanidine hydrochloride solution (prepared with pH 8.0, 50mM Tris HCl solution, and 1% by volume of protease inhibitor P8340, Sigma-Aldrich) to the precipitate, homogenize, and shake to mix at room temperature for 3-4 hours. Centrifuge at 100,000g, 4℃ for 60 minutes, and collect the supernatant. Then, dilute with BSAT-DPBS solution (0.2g / L KCl, 0.2g / L KH₂PO₄, 8.0g / L NaCl, 1.150g / L Na₂HPO₄, 5% BSA, 0.03% HCl). Tween-20 (pH 7.4, deionized water) was diluted 1:100 by volume, and the samples were stored at -80°C. Before starting the ELISA assay, the samples were thawed on ice and diluted 1:5 by volume with RIPA or BSAT-DPBS solution for ELISA detection. A standard curve was prepared simultaneously using standards for ELISA detection.

[0085] 3. ELISA detection of Aβ

[0086] (1) Preparation of Aβ1-40 and Aβ1-42 standards: Add 55mM NaHCO3 (pH 9.0) to the standards to prepare a 100ng / mL stock solution. Then, dilute the stock solution to different concentrations (1000pg / mL, 500pg / mL, 250pg / mL, 125pg / mL, 62.5pg / mL, 31.25pg / mL, 15.63pg / mL, and 7.81pg / mL) using standard diluent and aliquot into containers. Store at -80℃. Before use, thaw the sample on ice and add an equal volume of sample diluent (RIPA or BSAT-DPBS solution) to obtain the standard test sample.

[0087] (2) Add 50 μL of the standard test sample and the sample to the 96-well plate provided by the kit, and then immediately add 50 μL of the detection antibody provided by the kit. Incubate overnight on a shaker at 4°C.

[0088] (3) On the second day, take out the 96-well plate, discard the supernatant, dilute the washing buffer (25×) provided by the kit with deionized water, add 250 μL of washing buffer to each well, shake for 30 seconds, discard the washing buffer, drain on absorbent paper, and wash 5 times.

[0089] (4) Prepare rabbit antibody (1:100) for detection using HRP dilution buffer. Add 100 μL to each well, shake at room temperature for 30 minutes, discard the solution, and wash 5 times.

[0090] (5) Add 100 μL of colorimetric solution to each well and incubate at room temperature in the dark for 30 minutes.

[0091] (6) Add 100 μL of stop solution to terminate the colorimetric reaction, measure the optical density at a wavelength of 450 nm, calculate the standard curve according to the standard provided in the kit, and calculate the content of Aβ in the sample.

[0092] Experimental results:

[0093] Enzyme-linked immunosorbent assay (ELISA) of Aβ40 and Aβ42 revealed no significant difference in soluble Aβ40 between APP / PS1 / CHOP+ / - and APP / PS1 / CHOP- / - mice and APP / PS1 / CHOP- / - mice, while soluble Aβ42 was reduced in the hippocampus. Figure 5 A), but insoluble Aβ40 and Aβ42 were significantly reduced in both the cerebral cortex and hippocampus (A), but insoluble Aβ40 and Aβ42 were significantly reduced. Figure 5(B) Currently, it is believed that soluble Aβ mainly reflects the levels of monomers and oligomers, while insoluble Aβ represents the level of β-amyloid protein deposition. This result indicates that knocking down / knockout CHOP in AD mice significantly reduces Aβ amyloid protein deposition in the brains of AD mice.

[0094] Example 4: CHOP knockout reduces microglia activation in the cerebral cortex and hippocampus of AD mice.

[0095] Experimental methods:

[0096] 1. Sampling, fixation, and sectioning of brain tissue from transgenic mice

[0097] Five to seven 9-month-old mice from each group were used in the experiment. The mice were first anesthetized, and after deep anesthesia, the thoracic cavity was quickly opened to expose the heart. The heart was perfused with 50 mL of 0.9% sodium chloride solution, followed by 100 mL of fixative (4% paraformaldehyde solution). Brain tissue was dissected and placed in the fixative for 24 hours, then replaced with 30% sucrose solution. After the brain tissue settled, it was frozen sectioned using a sliding microtome (Lecia). Five brain slices with a bregma of -1.46 to -3.08 were selected for staining and quantification, with a section thickness of 25 μm.

[0098] 2. IbaI Immunohistochemical Staining Method

[0099] (1) Treat with 3% hydrogen peroxide solution (PBS: methanol: 30% hydrogen peroxide = 5:4:1) at room temperature for 30 minutes. Wash 3 times with phosphate buffer, 10 minutes each time.

[0100] (2) Treat with 0.1% v / v Triton-X 100 solution at room temperature for 10 minutes. Wash three times with phosphate buffer for 10 minutes each time.

[0101] (3) Block with antibody blocking solution (1% bovine serum albumin solution) at room temperature for 1 hour.

[0102] (4) Transfer the sections to the primary antibody solution (1% bovine serum albumin solution), dilute the IbaI antibody at a volume ratio of 1:1000, and incubate overnight at 4°C. Wash three times with phosphate buffer the next day, 10 minutes each time.

[0103] (5) Transfer the sections into the secondary antibody solution (goat anti-rabbit, 1:500) and incubate at room temperature for 2 hours. Wash 3 times with phosphate buffer, 10 minutes each time.

[0104] (6) Transfer the slices into horseradish peroxidase-labeled streptavidin (HRP, 1:1000) solution and incubate at room temperature for 2 hours. Wash three times with phosphate buffer for 10 minutes each time.

[0105] (7) 0.05% DAB phosphate buffer (a solution of 3,3'-diaminobenzidine dissolved in phosphate buffer, 25 mg / mL stock solution, diluted 1:50 before use), add hydrogen peroxide to a final concentration of 0.03%, and allow for appropriate color development time. Wash 3 times with phosphate buffer, 10 minutes each time;

[0106] (8) After mounting and drying, the slides were dehydrated and made transparent, then sealed with neutral resin. The slides were observed and photographed under a microscope (Olympus). The experimental results were then edited using Photoshop 9.0 software to obtain the images.

[0107] 3. Quantitative analysis of IbaI positive signal: Five consecutive brain slices from the same location in the cerebral cortex and hippocampus were selected at 40x magnification and quantitatively analyzed using ImageJ software. The intensity of the immunopositive signal per unit area in the cerebral cortex and hippocampus was measured for statistical analysis.

[0108] Experimental results:

[0109] Significant microglial activation was observed in AD mice, while the number of activated microglia was significantly reduced in APP / PS1 / CHOP+ / - and APP / PS1 / CHOP- / - mice compared to APP / PS1 mice (see [link to APP / PS1 mice]). Figure 6 (A and C in the text). Simultaneously, co-labeling with IbaI and ThioS fluorescent staining revealed that IbaI-positive microglia aggregated around ThioS-labeled β-amyloid protein (A and C in the text). Figure 6 (B in the text). This result indicates that CHOP knockout can reduce β-amyloid protein while simultaneously reducing microglial activation.

[0110] Example 5: CHOP knockout improves spatial learning and memory abilities in AD mice

[0111] Experimental methods:

[0112] The Morris water maze experiment is primarily used to test an animal's spatial learning and memory abilities. The specific method is as follows: A circular basin with a diameter of 120 cm and a height of 50 cm is filled with water to a depth of 30 cm, and the basin's horizontal plane is divided into four equal quadrants. White dye is then added to the basin to make the water cloudy, and the water temperature is controlled at 22±1℃. A platform with a diameter of 10 cm is then placed in one of the divided quadrants, with the platform surface 2 cm underwater. Throughout the experiment, the platform remains in the same position, and other experimental conditions are kept constant. Starting in one quadrant, the mouse is gently placed into the water facing the basin wall, and allowed to find the underwater platform within 60 seconds; the time taken is recorded. Once the mouse climbs onto the platform, it is allowed to remain on it for 10 seconds, then removed and placed in a separate cage to rest for approximately 60 minutes before moving to the next quadrant. If the mouse fails to find the platform within the specified time, it is placed on the platform and artificially allowed to remain there for 10 seconds. This process is repeated for the remaining three quadrants for five consecutive days. The average of the four training latency periods for each mouse was used as the learning data for that day. After five days of training, the underwater platform was removed, the mice were placed back in, and the time the mice spent in the quadrant of the platform and the number of times they crossed the platform were recorded within 60 seconds.

[0113] Experimental results:

[0114] Alzheimer's disease (AD) patients exhibit cognitive decline and memory loss, and a similar decline in spatial learning and memory abilities is observed in APP / PS1 mice. This behavioral abnormality can be verified using the Morris water maze test. In a Morris water maze test on 12-month-old mice, APP / PS1 mice, after 5 days of learning, showed that they took longer to find the platform and swam farther than other groups, indicating poorer spatial learning ability. The APP / PS1 / CHOP+ / - and APP / PS1 / CHOP- / - groups showed improvement compared to the APP / PS1 group. Figure 7 After the platform was removed, the APP / PS1 mice spent significantly less time in the platform quadrant and crossed the platform fewer times than the WT mice. The APP / PS1 / CHOP+ / - and APP / PS1 / CHOP- / - groups showed significant improvements compared to the APP / PS1 group. Figure 8 This experiment showed that APP / PS1 / CHOP+ / - and APP / PS1 / CHOP- / - mice had better learning and memory abilities regarding platform locations than APP / PS1 mice, indicating that CHOP knockdown / knockout can improve the learning and memory abilities of APP / PS1 mice.

[0115] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of CHOP gene in preparation of a medicament for treating Alzheimer's disease.

2. Use according to claim 1, characterized in that: The application is the use of a preparation for knocking out or knocking down CHOP gene in preparation of a medicament for treating Alzheimer's disease.

3. Use according to claim 2, characterized in that: The preparation for knocking out or knocking down CHOP gene includes small interfering RNA, gene editing molecules or CHOP antibodies.

4. The use according to any one of claims 1-3, wherein: The medicament for Alzheimer's disease can improve AD symptoms such as amyloid beta deposition, improve AD neuroinflammation, and improve learning and memory ability.

5. The use according to claim 4, wherein: The amyloid beta is insoluble amyloid beta in the cerebral cortex and hippocampus.

6. The use according to claim 4, wherein: The improvement of AD neuroinflammation is achieved by reducing microglial cell activation.

7. The use according to claim 6, wherein: The microglial cells are in the cerebral cortex and hippocampus.

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